RAPD, RFLP

Principle, working, application

1. RESTRICTION FRAGMENT LENGTH POLIMORPHISM & RANDOM AMPLIFIED POLYMORPHIC DNA neethu asokan

2. RFLP Definition The variation in the restriction DNA fragment lengths between individuals of a species is called restriction fragment length polymorphism ( RFLP ). Laboratory technique to analyze and compare DNAs of two or more individuals of a species or of different species. Genetic structure of all the individuals of a species is same, but at DNA level there are so many single base variations between the individuals due to point mutation. If a single base is altered due to mutation restriction enzymes never cut at the target sites. neethu asokan

3. HISTORY Shortly after Kary Mullis invented the Polymerase Chain Reaction (PCR) it was realized that short primers would bind to several locations in a genome and thus could produce multiple fragments. Williams et al. (1990) developed Random Amplified Polymorphic DNA (RAPD) a technique using very short 10 base primers to generate random fragments from template DNAs. RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprint. neethu asokan

4. RFLP TECHNIQUES neethu asokan

5. The RFLP involves the following steps: Sample collection Isolation of DNA Restriction digestion Electrophoresis Blotting of DNA Making genomic DNA probes Nucleic acid hybridization Autoradiography neethu asokan

6. Sample collection Tissues or cells of individuals are collected to extract their DNA. The samples are collected separately. Isolation of DNA P reparation of cell extract lysozyme - digestion of polymeric compounds EDTA - it removes Mg+ in the cell envelope SDS - removes the lipids of the cell wall. Purification of DNA DNA isolation neethu asokan

7. Restriction digestion Genomic DNA of each sample is cut with a restriction enzyme separately to generate variable lengths of DNA fragments. Restriction enzyme such as E.coR1, Hind ІІІ , Pst1 . The restriction digestion is divided into two half is used for DNA detection and the other is used for probe making. . neethu asokan

8. Electrophoresis The digested genomic DNA of all the samples are loaded into separate wells in A garose or Polyacrylamide gel and are subjected to electrophoresis. The DNA fragments get separated according to their size by using molecular weight markers neethu asokan

9. Blotting of DNA Southern blotting neethu asokan

10. Each sample is electrophoresed to separate the DNA fragments. Fragments of 0.5-2 kb are extracted from the gel and cloned in pUC21 vector to construct rDNAs. These rDNAs are amplified by introducing them into bacterial host and the amplified rDNAs are reisolated. Restriction digestion & electrophoresis. Radiolabelling by Nick translation or End labelling Genomic probes are formed. Making genomic DNA probes neethu asokan

11. Nucleic acid hybridization neethu asokan

12. Autoradiography neethu asokan

13. RFLPs are considered as the first class genetic markers to construct high resolution linkage maps of chromosomes. Used to construct chromosome map of human being, rice, wheat, maize, and microbes. Single gene diseases in man, plants and animals can be identified with RFLP markers. Monitoring inheritance of agronomic traits Diagnostic in genetically inherited disease Pedigree analysis, Forensic typing - Parentage analysis Identifying hybrids Species level relationship Also in some case at higher level relationship Applications of RFLP neethu asokan

14. Advantages Fingerprinting technique replacing RFLP Highly polymorphic High reproducibility Identify through absent or present of fragment Characters can be increased by changing the RE and nucleotide at selective primers Codominantly inherited. neethu asokan

15. Disadvantages Dominant – lose the codominant character Homology – ability to differentiate different fragment with similar size Mutation rate – high homoplasy – High levels of variation - similarity between two taxa are low , so both character and distance measures and tree reconstruction programmes are increasingly inaccurate – if levels of variation are high - Homoplasy Scoring - bias neethu asokan

16. Definition The RAPD is a PCR based method to detect variations between individuals of a species by selective amplification of some polymorphic sequences in their genomes. Developed by J.G.K.Williams et.al. in 1991 . Only least number of DNA fragments are considered for RAPD analysis. The set of DNAs generated by the random PCR is called RAPD. The RAPDs of one individual is differ from the RAPDs of other individuals both in number and size of DNA fragments. RAPD neethu asokan

17. Components of a PCR and RAPD Reactions PCR 1. Buffer (containing Mg ++ ) 2. Template DNA 3. 2 Primers that flank the fragment of DNA to be amplified 4. dNTPs 5. Taq DNA Polymerase (or another thermally stable DNA polymerase) RAPD Buffer (containing Mg ++ ) - usually high Mg ++ concentrations are used lowering annealing stringency 2 . Template DNA 3. 1 short primer (10 bases) not known to anneal to any specific part of the template DNA 4. dNTPs 5. Taq DNA Polymerase (or another thermally stable DNA polymerase) neethu asokan

18. Major steps in RAPD analysis Sample collection DNA isolation PCR Each genomic DNA is separately treated with T aq DNA polymerase, a primer, dATP ,dTTP, dGTP, dCTP, and polymerization buffer. All these reaction mixture are kept separately in a PCR equipment. subjected t o denaturation – 94 ° c for 1minute primer annealing -36 ° c for 2minutes polymerization – 72 ° c for 1.5minutes. neethu asokan

19. During the primer annealing, the primer molecules get bind with the polymorphic sequences found here and there throughout the genome by complimentary base pairing. RAPD Method neethu asokan

20. Provides 3’-OH for synthesis of new strands by polymerase activity during polymerization reaction. As a result of PCR amplification, each tube containing RAPD fragments. DNA fragments has a flanking primer at its 5’ end. Electrophoresis. Examined under the UV light illuminator and photographed. Light bands of the markers shows the molecular weights of each individual in different lanes neethu asokan

21. Uses of RAPD RAPDs are used as genetic markers for constructing genetic maps of higher organisms. Helps to identify genes of high economic value through comparison of RAPD fragments. Distinguishes one individuals from others of a species as fingerprinting analysis Determine specific genes in chromosomes It has become widely used in the study of Genetic diversity/polymorphism , Germplasm characterization , Genetic structure of populations, Domestication, neethu asokan

22. Detection of somoclonal variation, Cultivar identification , Hybrid purity , Genome mapping , Developing genetic markers linked to a trait in question, Population and evolutionary genetics, Plant and animal breeding, Animal-plant-microbe interactions , Pesticide/Herbicide resistance , neethu asokan

23. Advantages of RAPD No need for species specific probes in RAPD Probes prepared for one species may also used for other species Quick method Can be performed in crude DNA samples also Requires only a small amount of samples Doesn’t require radioactive probes and hybridization. It detects dominant variations in the genome neethu asokan

24. Disadvantages Profiling is dependent on the reaction conditions Profiles are not able to distinguish heterozygous from homozygous individuals- dominant marker Lack of a prior knowledge on the identity of the amplification products. Problems with reproducibility ( sensitive to changes in the quality of DNA, PCR components and PCR conditions). Problems of co-migration (do equal-sized bands correspond to the same homologous DNA fragment). Gel electrophoresis can separate DNA quantitatively, cannot separate equal-sized fragments qualitatively (i.e. according to base sequence). neethu asokan

25. References N.Senthikumar & G.Gurusubramanian , Random amplified polymorphic DNA (RAPD) markers and its applications 2011, www.sciencevision.in Timothy G. Standish, Ph. D . , Random Amplified Polymorphic DNA , (ppt). M.O . Dayhoff and R.V . ECK, Molecular techniques,(ppt). Biotechnology , V.Kumaresan, Saras publications. Biotechnology, U.Sathyanarayana. Gene Biotechnology, S.Jogdand. neethu asokan